Method for transferring material in a cell system

ABSTRACT

Described is a method for method for transferring material through the membrane of at least one cell, wherein the transfer is carried out in the presence of trehalose. This method is applicable in particular in the field of genetic engineering and biotechnology.

[0001] The present invention relates to a method for transferringmaterial through the membrane of at least one cell, as well as to theapplication of this method in genetic engineering, biotechnology andhybridoma technology.

[0002] Over the past few years, methods for transferring biologicalmaterials through the membrane of a cell have increasingly gained inimportance. In these methods, membrane-impermeable molecules aretransferred through pores that have formed in the membrane as a resultof extraneous forces. These methods are associated with a decisiveadvantage in that there is no need for the use of any vehicles.

[0003] Reversible membrane permeabilization through an electrical field,or alternatively, electroporation, has for some time been an establishedmethod of taking up free DNA, for example in eukaryotes. In thisprocess, eukaryotes, in the presence of DNA, are exposed to ahigh-strength electrical field. However, only very little is known aboutthe mechanism of DNA uptake during electroporation. It is generallyassumed that due to the electric shock, pores form temporarily in thecell membrane, and the DNA, after contact with the lipid bi-layer of thecell membrane, is taken up into the cell.

[0004] While in electroporation, material is imported to the cell fromthe exterior, electrofusion differs in that at least two cells fuse.Electrofusion takes place by means of electrical pulses in two stages.In the first step, the cells to be fused are subjected to an alternatingelectrical field in which they are mutually attracted to each other as aresult of dielectrophoresis. Conductivity of the medium should be as lowas possible. In the second step, electrofusion is triggered by veryshort electrical direct-current pulses. This leads to interaction ofmembrane parts which leads to fusion. With this method it is possible,for example, to fuse protoplasts. It is also possible to produce hybridsof animal cells, such as hybridoma cells, as well as yeasts.

[0005] It has thus been known for some time to cause transfer ofmaterial into a cell in that the cells are subjected to irradiationtreatment for the purpose of permeabilization. In this process, thecells are for example subjected to laser irradiation, after which thematerial can then diffuse through the cell membrane.

[0006] Methods have thus already been used in which, for the purpose ofpermeabilization, the membranes of cells are treated with chemicalsubstances. Such substances include pore-forming and/ordiffusion-promoting peptide antibiotics and depsipeptide antibiotics,such as valinomycin, and detergents, such as sodium dodecyl sulfate.

[0007] The methods described above are based on the followingfundamental principle: local apertures in the cell membrane result fromenergy-intensive electrical, electromagnetic, or mechanical forces suchas current pulses, irradiation, ultrasound and pressure, and chemicaltreatment. Submicroscopic holes or pores occur to form. Such treatmentmakes possible introducing the biological material from the exterior,or, between cells, if two cells are to be fused. After diminishedintensity of these forces acting from the exterior, the pores in themembrane close up, and the material remains in the cell.

[0008] However, it has been shown that in this method, the fraction ofsurviving cells, i.e. intact cells which have been reversiblypermeabilized, is often exceeded by the fraction of dead cells. This isdue to the permeabilized cells being unable to completely re-close thepores after a reduction in the intensity of the external forces. Thisthen leads to the loss of important cell functions so that the cell isno longer able to maintain its metabolism, a situation which finallyleads to the death of the cell. For this reason, it was quite normal inthe method according to the state of the art, to have to accept afraction of dead cells, a factor which had a significant negative effecton the efficiency of this method.

[0009] It is thus the object of the present invention to provide amethod for transferring material through the membranes of cells, bywhich a high degree of reversibly permeabilized, so-called survivingcells is achieved, and thus the fraction of dead cells is drasticallyreduced. An increase in the fraction of surviving cells should also berealized in the event of working with more stringent reactionconditions, for example increased field strengths.

[0010] This object is solved by the method according to claim 1. Thesub-claims relate to preferred embodiments of the method according tothe invention.

[0011] Furthermore, the claims describe particular applications of themethod according to the invention.

[0012] The present invention relates to a method for transferringmaterial through the membrane of at least one cell, in an aqueous mediumwhich is characterized in that the transfer is carried out in thepresence of trehalose.

[0013] The method according to the invention is explained in more detailwith reference to the accompanying figures. The following are shown:

[0014]FIG. 1—a graphic representation showing the propidium iodideuptake and the survival rate (% reversibly permeabilized cells)depending on the field strength in the hypo-osmolar medium;

[0015]FIG. 2—a graphic representation showing the “pulse efficiency” asa measure of the yield, depending on the field strength in thehypo-osmolar medium;

[0016]FIG. 3—a graphic representation showing the propidium iodideuptake and the survival rate (% reversibly permeabilized cells)depending on the field strength in the iso-osmolar medium; and

[0017]FIG. 4—a graphic representation showing the “pulse efficiency” asa measure of the yield, depending on the field strength in theiso-osmolar medium.

[0018] According to the invention it has been shown that the survivalrate of reversibly permeabilized cells is drastically increased iftrehalose is added to the aqueous working medium. Presumably, thetrehalose has a membrane-stabilizing and membrane-healing effect afterthe closure of the pores following introduction of the biologicalmaterial. Consequently, the permeabilized cells survive and there isthus no danger of cell functions dying due to the loss of cell fluidsand cell organelles.

[0019] The use of trehalose in methods of the type described here hasnot been known up to now. The literature merely indicates that trehalosemakes a contribution to the cryoconservation of mammalian cells (NatureBiotechnology, vol. 18, February 2000) and to sustaining intact humancells without the presence of water (Nature Biotechnology, vol. 18,February 2000).

[0020] In principle, any trehalose which is dissolved in the respectiveworking buffers is suitable. Trehalose is a disaccharide which occursnaturally; in one case it is also made synthetically. α,α-trehalose isthe best-known trehalose and the one that most frequently occursnaturally. α,β-trehalose also occurs naturally; it has been found to bepresent in honey. β,β-trehalose is only available synthetically.Preferably, α,α-trehalose is added to the working buffer fortransferring material.

[0021] The method according to the invention is preferably suitable forthe transfer of material in which at least one cell is involved which isreversibly permeabilized, or in which at least two cells which adhere toeach other are involved which are permeabilized and which practicallyexchange material between each other. In this case it is possible thatat least two cells fuse. There is also a further variant where severalcells fuse, quasi by forming a string of pearls.

[0022] Normally, transfer of material into the cell is through a localaperture or apertures in the membrane of the cell or the cells. This iscalled permeabilization of the membrane. Presumably material istransferred through part of the apertures while the other part of theapertures remains without material passage. Having taken up thematerial, these apertures too, have to close. It is especially in thecontext of this process that trehalose has proven to be particularlyadvantageous as a membrane-healing additive.

[0023] As a rule, permeabilization of the membrane can take place byapplying an electrical field, by irradiation, or by chemical treatment.The actual method selected essentially depends on the type of cells tobe transformed and the type of material to be transferred.

[0024] Methods including electrotransfection, electroporation, orelectrofusion, either in macroscopic devices or inmicrosystems/microstructures, are suitable for electricalpermeabilization. These are established methods which have beensuccessfully in use for some years in gene technology. In particular inthe case of electroporation it is often necessary to work with highfield strengths. However, this has traditionally been associated with aproblem in that the cells subsequently died, i.e. that they had beenirreversibly permeabilized. This led to considerable reductions inyield. However, if, according to the invention, trehalose is added tothe electroporation buffer, it is possible to obtain reversiblypermeabilized living cells, even at high field strengths. This factorrenders the method according to the invention far more economical.

[0025] Thus, permeabilization by way of UV irradiation or laserirradiation is also possible. This type of irradiation is advantageouswhen due to the cells used and due to the material to be transferred,electrical treatment is not indicated.

[0026] Thus, chemical treatment for permeabilization is recommended onlywhen it is not advisable to carry out transfer in the electrical fieldor transfer by way of irradiation. If chemical treatment is to be used,then, for permeabilization, the cells are to be treated withantibiotics, detergents, etc.

[0027] With the method according to the invention, any suitablebiological materials can be transferred, including: xenomolecules, DNAand RNA, plasmids, chromosomes, parts of chromosomes as well asartificial chromosomes, proteins and glycoproteins, cells, parts ofcells, and cell organelles, or low-molecular foreign matters.

[0028] The biological material can be trehalose or atrehalose/saccharose mixture. In this way it is possible to introducetrehalose or the trehalose/saccharose mixture as an intracellularcryoprotectant or protectant against desiccation.

[0029] As far as the cells used during transfer are concerned, themethod according to the invention is not subject to any limitations. Itis possible to use natural cells or membrane-enveloped vesicles for thetransfer of material. Natural or artificial vesicles, liposomes andmicelles are examples of membrane-enveloped vesicles.

[0030] In the case of natural cells, according to the invention it ispossible to permeabilize and transform prokaryotic and eukaryotic cells.Bacteria, blue algae and archae-bacteria are examples of prokaryoticcells. Eukaryotic cells can have their origins in protozoa, plants(including algae), fungi (including yeasts), animals or humans.

[0031] For electroporation and electrofusion, it is possible to work inthe iso-osmolar medium or in the hypo-osmolar medium. In animal cells,the hypo-osmolar medium has an osmolarity of 75 to 250 mOsm and is thusnonphysiological. The osmolarity of an iso-osmolar medium is approx. 300mOsm, with the medium corresponding to the physiological environment. Incontrast, in the case of plant cells, the osmolarity of an iso-osmolarmedium is approx. 500 mOsm. The hypo-osmolar medium ranges from approx.400 to 450 mOsm.

[0032] It has been found that the protective effect of trehalose becomesevident in particular in the hypo-osmolar medium.

[0033] It has been shown-that the concentration of trehalose in anaqueous medium, for example in the case of electrical treatment, shouldbe within the range of 1 to 200 mM. It has been found that trehaloseincreases the fraction of surviving cells after pulse application, withoptimal yield occurring at approx. 30 to 50 mM which is not increased byfurther increasing the concentration level. Therefore, a concentrationrange of approximately 30 to 50 mM is preferred for pulse application.

[0034] It has been shown that a further embodiment of the methodaccording to the invention brings about increased yield of reversiblypermeabilized cells even if a mixture of trehalose and saccharose isadded to the working buffer. The ratio of trehalose to saccharose rangesfrom 1:2 to 1:10. The concentration in the mixture ranges from 200 to300 mM.

[0035] The present method is eminently suitable for introducingbiological material into a cell, or for transfer between at least twocells. The method can therefore be applied in practically all areas ofbiotechnology, genetic engineering and microsystem technology. Inparticular, it is especially suited to hybridoma technology, for examplewhere elecrofusion is used. Furthermore, plant protoplasts can easily befused with the method according to the invention.

[0036] As a result of the presence of trehalose in the working medium,the method according to the invention has many advantages. These are dueto the fact that trehalose has a protective effect on the cells to betreated. Trehalose stabilizes the cell membrane and causes fast healingof the pores, for example following the uptake of foreign material andweakening of the external forces. In particular in the case ofelectroporation it has been found that the protective effect oftrehalose is very pronounced at high field strengths, while at the sametime hardly being affected by the pulse duration. It has been shown thatthe protective effect of trehalose is stronger in the hypotonic pulsemedium than it is in the isotonic pulse medium. The effect of trehaloseis somewhat more pronounced in a poorly conductive medium than it is ina stronger conducting medium. These advantageous protective propertiesof trehalose are in particular highly beneficial if work is carried outat stringent pulse conditions, such as poor conductivity, hypotonicstress, or high field strength.

[0037] Below, the method according to the invention is explained in moredetail by means of examples.

EXAMPLES Example 1

[0038] Electroporation of Cells with and without Trehalose in aHypo-osmolar Working Medium.

[0039] A phosphate buffer with 1.15 mM K₂HPO₄/KH₂PO₄ buffer, pH 7.2, wasused as a pulse medium. KCl at a concentration of 10 mM (α=1.5−1.6mS/cm) was added as a conducting salt. After this, trehalose at therespective concentration was added to the pulse medium. Osmolarity wasadjusted to 100 mOsm by the addition of inositol, so as to obtain ahypo-osmolar solution.

[0040] Jurkat cells that are cells of a human T-lymphocytes line wereused. 40 μg/ml propidium iodide, which is a membrane-impermeable DNAdye, was added as the material to be transferred.

[0041] Pulse application took place after 10 minutes of incubation priorto pulse application, in the pulse medium at room temperature (celldensity: 2-3×10⁶ cells/ml). Pulse duration was 20 μs.

[0042] Electroporation took place in an Eppendorf multiporator. Afterpulse application, the pores were left to reseal for 10 min at roomtemperature.

[0043] Electropermeabilisation took place at the following trehaloseconcentrations: 0 mM and 40 mM (α,α-trehalose).

[0044] Pulsing takes place at 4° C. or at room temperature. This can besingle pulsing; however, multiple pulsing involving up to 3 pulses canat times be advantageous.

[0045] The results are shown in FIGS. 1 and 2.

[0046]FIG. 1 diagrammatically shows the uptake of propidium iodide orthe survival rate of the electropermeabilized cells depending on thefield strength. Poration without trehalose is shown by squares.Preparation with 40 mM Trehalose is shown by triangles. Outline symbolsdesignate the survival rate (percentage of reversibly-permeabilizedcells) while solid symbols designate the propidium iodide uptake intothe cell.

[0047] As shown in FIG. 1, in a pulse medium without trehalose, cellssustain irreversible damage at field strengths from 1.5 kV/cm onwards,and die-off as a result of loss of cell functions (outline squares). Incontrast, in a pulse medium in 40 mM trehalose, there is only arelatively small percentage of dead cells (outline triangles), even atvery high field strengths up to 2.5 kV/cm. This is an indication thatthe cells were reversibly permeabilized and have thus remained viable.Propidium iodide uptake is only slightly influenced by trehalose.

[0048]FIG. 2 depicts an investigation of the pulse efficiency whichrepresents the product of propidium iodide uptake and survival rate, asa measure of the yield obtained from electroporation, depending on thefield strength. It is evident that with 40 mM trehalose in the pulsemedium (triangles), these values rise rapidly as the field strengthincreases, and are significantly above the values of a control sample(squares) which was not treated with trehalose. This significantincrease in yield is also due to a greatly improved survival rate in thepresence of 40 mM trehalose.

Example 2

[0049] Electroporation of Cells with and without Trehalose in anIso-osmolar Working Medium.

[0050] Essentially, the same experimental conditions as in Example 1applied, except that the osmolarity of the pulse medium was adjusted toiso-osmolar conditions by adding inositol to 290 mOsm.

[0051] Again, α,α-trehalose was added in the following concentrations: 0mM and 40 mM.

[0052] The results are shown in FIGS. 3 and 4.

[0053]FIG. 3 shows the dependence of the survival rate and the propidiumiodide uptake in relation to the field strength. Outline symbolsdesignate the survival rate, while solid symbols designate the propidiumiodide uptake.

[0054] In the iso-molar pulse medium, the survival rate in the case ofuntreated cells (0 mM, outline squares) is many times less than in thecase of cells treated with 40 mM trehalose (outline triangles), aboveall at high field strength. In contrast, propidium iodide uptake variesless markedly. It is important to note that from a field strength of 3kV/cm onwards, there is a drastic die-off of cells if there is notrehalose in the pulse medium.

[0055]FIG. 4 shows the pulse efficiency dependent on the field strengthfor the present pulse media (0 to 40 mM trehalose). As the fieldstrength increases, a gradual increase in the pulse efficiency isevident in the pulse medium containing trehalose (triangles), whilewithout trehalose (squares) in the pulse medium at high field strength,a drastic reduction in yield is evident. Here again, trehalose displaysits protective effect in stringent pulse conditions.

1. A method for transferring material through the membrane of at leastone cell in an aqueous medium, wherein transfer takes place in thepresence of trehalose.
 2. The method according to claim 1, wherein thattransfer of the material takes place from the exterior into the cell, orthe material is transferred between at least two cells.
 3. The methodaccording to claim 1 or 2, wherein that transfer takes place by way ofpermeabilization of the membrane of the cell or cells.
 4. The methodaccording to claim 3, wherein that permeabilization of the membranetakes place by applying an electrical field, by irradiation or chemicaltreatment.
 5. The method according to claim 4, wherein forpermeabilization, electrical methods are applied, includingelectrotransfection, electroporation or electrofusion, either inmacroscopic devices or in microsystems/microstructures.
 6. The methodaccording to claim 4, wherein the membrane is permeabilized by laserirradiation or ultrasound.
 7. The method according to claim 4, whereinthe membrane is permeabilized by treatment with antibiotics ordetergents.
 8. The method according to claim 1, wherein biologicalmaterial is used as material to be transferred.
 9. The method accordingto claim 8, wherein xenomolecules, DNA and RNA, plasmids, chromosomes,parts of chromosomes as well as artificial chromosomes, proteins andglycoproteins, cells, parts of cells and cell organelles, orlow-molecular foreign matter, trehalose or a trehalose/saccharosemixture are transferred as biological materials.
 10. The methodaccording to at least one of claims 1 to 9, wherein the cells used fortransfer are natural cells or membrane-enveloped vesicles.
 11. Themethod according to claim 10, wherein prokaryotic and eukaryotic cellsare used as natural cells.
 12. The method according to claim 11, whereinthe eukaryotic cells are of human, animal, or plant origin.
 13. Themethod according to at least one of claims 1 to 12, wherein theconcentration of trehalose in an aqueous medium is within the range of 1to 200 mM.
 14. The method according to claim 13, wherein the preferredconcentration is 30 to 50 mM.
 15. The method according to claim 1,wherein the concentration of trehalose is from 200 to 300 mM, whentrehalose is present in admixture with saccharose in a ratio of from 1:2to 1:10 (trehalose:saccharose).
 16. The method according to claim 1,wherein an iso-osmolar or hypo-osmolar medium is used as an aqueousmedium.
 17. The use of a method according to claim 1, for introducingbiological material into a cell.
 18. The use according to claim 17,wherein the biological material itself is trehalose or a mixture oftrehalose and saccharose.
 19. The use according to claim 16 in the fieldof genetic engineering, biotechnology, hybridoma technology, andmicrosystem technology.